Novel VHH-Based Tracers with Variable Plasma Half-Lives for Imaging of CAIX-Expressing Hypoxic Tumor Cells

Hypoxic areas are present in the majority of solid tumors, and hypoxia is associated with resistance to therapies and poor outcomes. A transmembrane protein that is upregulated by tumor cells that have adapted to hypoxic conditions is carbonic anhydrase IX (CAIX). Therefore, noninvasive imaging of CAIX could be of prognostic value, and it could steer treatment strategies. The aim of this study was to compare variants of CAIX-binding VHH B9, with and without a C-terminal albumin-binding domain with varying affinity (ABDlow and ABDhigh), for SPECT imaging of CAIX expression. The binding affinity and internalization of the various B9-variants were analyzed using SK-RC-52 cells. Biodistribution studies were performed in mice with subcutaneous SCCNij153 human head and neck cancer xenografts. Tracer uptake was determined by ex vivo radioactivity counting and visualized by SPECT/CT imaging. Furthermore, autoradiography images of tumor sections were spatially correlated with CAIX immunohistochemistry. B9-variants demonstrated a similar moderate affinity for CAIX in vitro. Maximal tumor uptake and acceptable tumor-to-blood ratios were found in the SCCNij153 model at 4 h post injection for [111In]In-DTPA-B9 (0.51 ± 0.08%ID/g and 8.1 ± 0.85, respectively), 24 h post injection for [111In]In-DTPA-B9-ABDlow (2.39 ± 0.44%ID/g and 3.66 ± 0.81, respectively) and at 72 h post injection for [111In]In-DTPA-B9-ABDhigh (8.7 ± 1.34%ID/g and 2.43 ± 0.15, respectively). An excess of unlabeled monoclonal anti-CAIX antibody efficiently inhibited tumor uptake of [111In]In-DTPA-B9, while only a partial reduction of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh uptake was found. Immunohistochemistry and autoradiography images showed colocalization of all B9-variants with CAIX expression; however, [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh also accumulated in non-CAIX expressing regions. Tumor uptake of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh, but not of [111In]In-DTPA-B9, could be visualized with SPECT/CT imaging. In conclusion, [111In]In-DTPA-B9 has a high affinity to CAIX and shows specific targeting to CAIX in head and neck cancer xenografts. The addition of ABD prolonged plasma half-life, increased tumor uptake, and enabled SPECT/CT imaging. This uptake was, however, partly CAIX- independent, precluding the ABD-tracers for use in hypoxia quantification in this tumor type.


■ INTRODUCTION
Hypoxia is a key feature of solid tumors. As it is closely associated with disease progression and therapy resistance, 1,2 identification of hypoxic subvolumes through noninvasive imaging could aid in steering cancer treatment. Therefore, the development of imaging probes detecting hypoxia is ongoing.
Traditional probes like [ 18 F]-FMISO, [ 18 F]-FAZA and [ 18 F]-HX4 accumulate in tissues with oxygen pressures below 10 mmHg at any point in time during circulation of the probe. 3−5 Therefore, they indicate diffusion-limited hypoxia and tumor areas with longer-lasting perfusion-limited hypoxia, while, for treatment resistance, especially the cells that have adapted to hypoxic conditions are of interest. Carbonic anhydrase IX (CAIX) is a transmembrane protein that is upregulated on tumor cells that have adapted to hypoxic conditions, 6,7 and consequently, imaging of CAIX is a potential strategy to visualize clinically relevant hypoxia.
The variable domains of heavy chain only antibodies (VHHs) are biomolecules with high potential for tumor imaging and therapy. 8−12 Due to their low molecular weight of about 15 kDa, VHHs are rapidly cleared from the blood through glomerular filtration, leading to high tumor-to-background ratios at early time points after injection. Furthermore, the small size of VHHs leads to efficient and homogeneous tissue penetration. 13,14 Though fast blood clearance is advantageous for image quality at early time points after injection, it can also limit the absolute uptake of the VHH in tumor lesions, which is problematic for imaging of low-abundant targets or in therapeutic applications, for which high absolute uptake of the drug is important. 15 To this end, strategies to increase plasma half-life have been applied to VHHs, such as the addition of polyethylene glycol (PEG), conjugation to the Fc domain of conventional antibodies, and engineering of multimeric or multivalent VHH compounds. 16−18 Another promising method to extend the plasma half-life of compounds is the addition of an albumin-binding domain (ABD). 19 Albumin is an abundant serum protein with a plasma half-life of 19 days, which is continuously recycled via neonatal F C receptor (FcRn)-mediated transcytosis. Association of the VHH with albumin will thus lead to prolonged serum residence through these mechanisms. 18,20 In previous studies, this strategy has been shown to increase plasma half-lives of other imaging and therapy tracers such as peptides, VHHs and affibodies. 21−30 Here, we used the previously selected VHH against CAIX. 31 The radiolabeled VHH B9 was further developed and characterized for nuclear imaging of CAIX. We fused B9 to albumin-binding domains with varying affinity (ABD low and ABD high ), 26 to assess the effect on the pharmacokinetics of the tracer and tumor uptake. We validated the potential of these tracers for nuclear imaging of endogenous hypoxic CAIXexpressing regions in a human head and neck squamous cell carcinoma xenograft model.

VHH Expression and Conjugation.
Anti-CAIX VHH B9 31 and B9 with a C-terminal albumin-binding domain (ABD) of streptococcal protein G 26 were used in this study. The VHHs were recloned to vectors that introduce a C-terminal cysteine, followed by the FLAG tag for B9 and the EPEA tag for B9-ABD high and B9-ABD low, and produced as described previously. 31 In short, bacteria were grown in Terrific Broth (TB, for B9) or 2× Trypton Yeast Extract medium (2×TY, B9-ABD low and B9-ABD high ) and VHH production was induced with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) overnight at 25°C . B9 was purified from the periplasmic fraction using a HiTrap protein A HP column (GE Healthcare, Chicago, IL, USA), and B9-ABD low and B9-ABD high were purified with a HiTrap protein A HP column and a CaptureSelect C-tag Prepacked Column (Thermo Scientific, Waltham, MA, USA). To reduce the cysteines, VHHs were incubated with Tris(2-carboxyethyl)phosphine hydrochloride (TCEP; Sigma-Aldrich, Saint Louis, MS, USA) at a molar ratio of 1:2.75 overnight at room temperature in PBS pH 7.5. Subsequently, VHHs were incubated with maleimide-DTPA (C-107; Chematech, Dijon, France) at a molar ratio of 1:5 in PBS pH 7.5 for 3 h at room temperature. The reaction mixture was dialyzed in 0.25 M ammonium acetate buffer, pH 5.5, to remove excess maleimide-DTPA.
Binding to Murine and Human Albumin. Human serum albumin (HSA) and mouse serum albumin (MSA) (Sigma-Aldrich BV, Zwijndrecht, The Netherlands) were coated on MaxiSorp plates (5 μg/well) and blocked with 1% BSA/PBS. Upon incubation with 0.5−1000 nM of each VHH in 1% BSA/ PBS for 2 h at room temperature, the unbound fraction was washed away, and the bound VHHs were detected with a combination of a rabbit anti-VHH antibody (clone QE19, QVQ B.V.) and a goat anti-Rabbit IRDye800CW (LI-COR Biosciences). The signal was detected using the Odyssey Infrared Imager (LI-COR Biosciences), and fluorescent intensities were plotted over VHH concentration. The binding affinity (K D ) was determined by a nonlinear regression curve fitting for one-site specific binding. In-DTPA-B9 high (7.1 μg, n = 3), respectively. For each tracer, 3 mice were injected with an excess of monoclonal anti-CAIX antibody girentuximab (1 mg/mouse) 24 h before tracer injection to determine CAIX-specificity of uptake. Mice were anesthetized with isoflurane, and the tumor area was scanned in the prone position using the 1.0 mm diameter multipinhole mouse collimator. SPECT scans were acquired for 120 min using 6 bed positions. After scanning, mice were injected intravenously with Hoechst 33342 (15 mg/kg, Sigma, Zwijndrecht, The Netherlands), and after 1 min, they were euthanized by cervical dislocation. Tissue samples and half of the tumors were used for radioactivity counting, and the other half of the tumors were snap-frozen in liquid nitrogen for immunohistochemistry and autoradiography as described.
Scans were reconstructed with 4 iterations and 16 subsets using an ordered-expectation maximization algorithm with a voxel size of 0.375 mm (MILabs reconstruction software). SPECT images were analyzed with Inveon Research Workplace software (version 3.0; Siemens Preclinical Solutions).
Autoradiography and CAIX Immunohistochemistry. Snap-frozen tumors were cut into 5 μm sections and mounted on poly L-lysine coated slides. These were exposed to phospholuminescence plates in a Fujifilm BAS cassette 2025 for 36 h (Fuji Photo Film), and scanned using a Fuji BAS-1900 II bioimaging analyzer at a pixel size of 25 μm × 25 μm. After fixation with cold acetone for 10 min at 4°C, immunohistochemical staining and analysis for CAIX and Hoechst was performed as described previously. 34 Intratumoral correlation analysis was performed as described by Huizing et al. 35 by resizing and aligning CAIX immunohistochemistry and autoradiography images. A parametric mapping technique was applied, and a correlation coefficient for colocalization of CAIX staining and autoradiography in viable tumor tissue was calculated.
Statistics. Graphpad Prism was used for statistical analyses. Statistical significance was determined with an unpaired t test or one-way ANOVA, and correlation coefficients were calculated    Table 2). The addition of ABD low and ABD high to B9 led to increased tumor uptake; however, enhanced blood retention led to lower tumor-to-blood ratios when compared to B9. Maximal tumor-to-muscle and acceptable tumor-to-blood ratios (>2 for application in  Table 2). Fusion of B9 to ABD high led to a strong decrease in kidney uptake, which was reflected in increased tumor-to-kidney ratios at all time points, while fusion to ABD low  (Figure 6c), heterogeneous uptake in the SCCNij153 xenograft could be visualized, which partly remained upon injection of an excess of girentuximab.
Signal of autoradiography in excised tumor sections correlated positively with CAIX immunohistochemistry for all B9-based tracers ( Supplementary Figure 5a−d). Importantly, these analyses were done on annotated vital tumor areas, excluding regions of keratinization in the tumor centers (marked as KE in Figure 6). The signal on autoradiography was efficiently blocked in CAIX positive regions but remained in the CAIX-negative keratinized regions for the blocked groups of B9-ABD low and B9-ABD high (Figure 6b,c).

■ DISCUSSION
Molecular imaging of hypoxic regions in solid tumors has the potential to steer treatment regimens. In the present study, we have developed and characterized novel radiotracers for SPECT  B9-variants were site-specifically equipped with maleimide-DTPA. We showed moderate binding affinity of conjugated B9  In-DTPA-B9-ABD high were found in the blood at 72 h, a later time point after injection could be more optimal for imaging, provided that tumor uptake of the tracer is retained. This would, however, eliminate the advantage of early imaging with VHHs when compared to imaging with monoclonal antibodies, which is also performed at 3−5 days post injection. 35 Importantly, the lower tumor accumulation of B9 when compared to that of labeled antibody girentixumab in other studies can be attributed to the 10-fold lower affinity and the inefficient internalization when compared to girentuximab. 36 Increased tumor uptake of both B9-ABD tracers could not efficiently be blocked by administration of unlabeled girentuximab, which binds a similar epitope of CAIX, indicating that the uptake is partly CAIX-independent. Autoradiography analysis indeed confirmed tracer uptake of ABD-conjugates in CAIXnegative keratinized tumor tissue, hypothetically due to increased residence of plasma proteins, possibly including albumin in this tissue type. Importantly, keratinization is a common histological feature of squamous cell carcinomas; 37 thus, aspecific uptake in these areas could restrict the application of these tracers in hypoxia quantification. Specific tumor accumulation using VHHs provided with an ABD might be possible for other tumor types lacking keratinized tissue, which remains to be investigated. Also, other groups have reported increased tumor uptake of peptide-, affibody-, and nanobodybased tracers conjugated to a variety of ABDs. In some studies, target specificity of the tracer was confirmed by lack of uptake in antigen-blocked groups or in nontarget expressing tumors, 29 while, in other studies, residual uptake in blocked groups or nontarget expressing tumors was observed. 28 Depending on the application, the current study underscores the importance of investigating the specificity of the tracer uptake.
Low absolute uptake of B9 precludes the use of this tracer for imaging of low-abundant targets such as CAIX in hypoxic tumors. The addition of ABD increases absolute but also nonspecific tumor uptake, also eliminating the possibility of using these constructs for precise localization and quantification of hypoxic tumor regions. Alternatively, other clearance affecting approaches could be used such as PEGylation, fusion to Fc domains, or multivalent constructs. 15 Aside from imaging, the addition of ABD could also be employed to increase absolute tumor uptake and decrease renal uptake of small tracers equipped with therapeutic radionuclides, thereby increasing therapeutic efficacy and decreasing nephrotoxicity. We observed a strong decrease in renal accumulation, especially for the ABD high conjugate. This might reflect the high affinity association of the tracer with plasma albumin that lasts for up to 72 h and induces increased hepatic excretion, while B9-ABD low has dissociated from plasma albumin within 24 h, and can be removed through glomerular filtration.
In recent years, multiple CAIX-targeting radiotracers have been studied. 38 An important advantage of VHHs as imaging tracers is their modular character. They can be site-specifically modified, thereby controlling target binding activity and biodistribution. Furthermore, they can be easily adjusted in size and valency and expressed in fusion with other proteins.

■ CONCLUSIONS
We demonstrated that the developed B9 tracers have a moderate affinity to CAIX in vitro and that they target head and neck cancer xenografts in vivo. This targeting was entirely CAIX specific for native B9, and although the addition of ABDs increased plasma residence and tumor uptake, tumor uptake of the ABD-modified VHH was partly CAIX-independent in keratinized areas. The addition of an ABD to B9, therefore, did not improve SPECT imaging contrast of CAIX-expressing regions for hypoxia quantification in head and neck cancer. This strategy could, however, be used to increase the absolute tumor uptake and to optimize tumor-to-kidney ratios for therapeutic applications of VHHs. ■ ASSOCIATED CONTENT
Uptake values and tumor-to-organ ratios of the various tracers in the biodistribution experiments, SPECT/CT images, autoradiography and immunohistochemistry figures of all animals included in the study, and the analyses of spatial correlation of autoradiography and CAIX expression on immunohistochemistry (PDF)